Figure 5. fard-1 overexpression is sufficient to extend lifespan by modulating ether lipid synthesis.

(A–B) Two independently generated fard-1 overexpression (fard-1 oe1 and fard-1 oe2) transgenic strains exhibit lifespan extension that is not further extended by concomitant phenformin treatment. (C) RNA interference (RNAi) knockdown of ads-1 fully suppresses fard-1(oe1) lifespan extension, indicating that the fard-1(oe)-mediated lifespan extension is dependent upon ether lipid synthesis. (D) qRT-PCR analysis of wild-type and fard-1(oe3) animals treated with vehicle or phenformin until adult day 1 reveals that both biguanide treatment and fard-1 exogenous overexpression results in an equivalent reduction of native fard-1 gene expression, as indicated by primers targeting the native 3’ UTR of fard-1, a sequence not represented in the fard-1 overexpression transgene (n=3 biological replicates). (E–G) RNAi of skn-1 (E), aak-2 (F), and daf-16 (G) suppress fard-1(oe1)-mediated lifespan extension. For (A–C) and (E–G), results are representative of two to three biological replicates. *, p<0.05; ***, p<0.001; ****, p<0.0001 by log-rank analysis. Note that (F–G) contain the same wild-type controls as they are visualized from the same replicate of the study. See also Figure 4—figure supplement 1 and Supplementary file 1 for tabular survival data and biological replicates. **, p<0.01 by two-way ANOVA followed by Tukey’s multiple comparisons test. (H) Worms overexpressing a backcrossed, integrated FARD-1 (fard-1 oe3) display a significant increase in 16:0 and 18:1 but not 18:0 alkenyl ether lipids by gas chromatography/mass spectrometry (GC/MS). (I) Comparison of the total fatty acid pool indicates that the polyunsaturated fatty acids 20:4 arachidonic acid (ARA) and 20:5 eicosapentaenoic acid (EPA) are significantly increased in fard-1 overexpressing (fard-1 oe3) worms vs. wild-type animal, while several isomethyl (iso) and cyclopropyl (cyclo) fatty acids change in opposing directions. For (H–I), n=3 biological replicates. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 by multiple t-tests (with multiple hypothesis correction by two-stage step-up method of Benjamini, Krieger, and Yekutieli).

Figure 5—figure supplement 1. fard-1 overexpression extends lifespan in a manner dependent upon ether lipid biosynthesis, and not apparently involving ferroptosis.

(A–B) RNA interference (RNAi) knockdown of fard-1 (A) and acl-7 (B) suppresses fard-1 overexpression(oe1)-associated lifespan extension. (C–E) Independent knockdown of glutathione peroxidases, gpx-1 (C), gpx-6 (D), or gpx-7 (E) by RNAi does not mitigate lifespan extension by integrated fard-1 overexpression (fard-1 oe3 and fard-1 oe4), as would be expected if fard-1 overexpression extended lifespan by lowering ferroptosis. gpx-1 knockdown unexpectedly extends lifespan in a non-additive manner with fard-1 (oe4). (F–H) Similarly, knockdown of gpx-1 (F), gpx-6 (G), or gpx-7 (H) by RNAi do not suppress phenformin-mediated lifespan extension. For (A–H), results are representative of two to three biological replicates. Note that (C–H) contain the same wild-type (wt) controls as they are visualized from the same replicate of the study. ***, p < 0.001; ****, p < 0.0001 by log-rank analysis. For tabular survival data and biological replicates, see also Supplementary file 1.